Monoamine neurotransmitters including histamine regulate multiple physiological processes, including gastric acid secretion in humans. In Drosophila, histamine mediates photoreceptor synaptic transmission. In both organisms, monoamine transmitters are released from secretory vesicles during exocytosis. Since transmitter synthesis occurs in the cytoplasm, exocytotic release requires active transport into the vesicle lumen. Specific vesicular monoamine transporters (VMATs) mediate this activity and include the products of two distinct human VMAT genes (VMAT1 in adrenal and VMAT2 in neurons), and one predicted gene in Drosophila. Previously, we have studied the regulation of rat VMAT2, and have identified a dileucine-like signal encoded in the carboxy terminus of VMATs that mediates efficient endocytosis. In addition we have identified two motifs that regulate transporter sorting to secretory vesicles. These include two acidic residues at the -4 and -5 position relative to the dileucine motif, and a distinct cluster of acidic residues at the extreme carboxy terminus of VMAT2. To initiate studies of VMAT in Drosophila, we recently cloned and sequenced cDNAs encoding Drosophila VMAT (dVMAT). Sequence analysis predicts at least two alternatively spliced dVMAT varaints (A and B) that contain different predicted carboxy-terminal cytoplasmic domains. dVMAT-A contains sequences strikingly similar to the dileucine motif and acidic cluster that regulates membrane trafficking of rat VMAT2. The other variant, dVMAT-B, lacks these signals. To test whether alternative splicing of membrane trafficking signals represents a new mechanism for regulating VMAT function, we will now determine the localization of the two variants invivo, and test the function of the potential dileucine motif in dVMAT-A. To determine how changes in dVMAT localization affect transmitter release invivo, we will also screen for dVMAT mutant flies. In future experiments, we will use a dVMAT mutant as a null background to express VMAT variants showing different patterns of intracellular trafficking. Using vision as a behavioral readout, we will determine how changes in dVMAT trafficking affect histamine storage and release. I will also use VMAT mutants in Drosophila as a sensitized background for future genetic screens to identify molecules that regulate VMAT function.